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. 2010 Jan 15;337(2):303-12.
doi: 10.1016/j.ydbio.2009.10.046. Epub 2009 Nov 6.

Hippo signaling regulates Yorkie nuclear localization and activity through 14-3-3 dependent and independent mechanisms

Fangfang Ren  1 , Lei Zhang, Jin Jiang
Affiliations

Hippo signaling regulates Yorkie nuclear localization and activity through 14-3-3 dependent and independent mechanisms

Fangfang Ren et al. Dev Biol. .

Abstract

The Hippo (Hpo) signaling pathway controls cell growth, proliferation and apoptosis in both Drosophila and vertebrates. In Drosophila, Hpo signaling regulates gene expression by inhibiting a transcription complex consisting of the transcriptional coactivator Yorkie (Yki) and the TEAD/TEF family of transcription factor Scalloped (Sd). Here we provide genetic evidence that both isoforms of 14-3-3, 14-3-3varepsilon and 14-3-3zeta, regulate Yki activity through modulating its subcellular localization. Inactivation of 14-3-3 by RNAi or genetic mutations enhanced whereas overexpression of 14-3-3 suppressed tissue overgrowth induced by Yki overexpression. Loss of 14-3-3 resulted in the accumulation of Yki in the nucleus. We found that regulation of Yki by 14-3-3 was mediated by phosphorylation of Yki at S168. In addition, we found that Hpo signaling also inhibited Yki nuclear localization and activity by phosphorylating Yki at S111 and S250, and this inhibition appears to be independent of 14-3-3. Finally, we provided evidence that Hpo signaling restricted Yki nuclear localization depending on CRM1-mediated nuclear export.

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Figures

Figure 1
Figure 1. Loss of function of either 14-3-3 isoform can enhance Yki-induced eye overgrowth
(A-L′) Side views (A-L) or dorsal views (A′-L′) of adult eyes of GMR-Gal4 (A and A′), GMR-Gal4 UAS-Yki (B and B′), GMR-Gal4 UAS-Yki; UAS-14-3-3εRNAi (C and C′), GMR-Gal4 UAS-Yki; UAS-14-3-3ζRNAi (D and D′), GMR-Gal4 UAS-Yki; UAS-14-3-3 εRNAi/UAS-HA-14-3-3ζ(E and E′), GMR-Gal4 UAS-Yki; UAS-14-3-3ε RNAi/UAS-HA-14-3-3ε(F and F′), GMR-Gal4 UAS-Yki; UAS-14-3-3ζ RNAi/UAS-HA-14-3-3ε(G and G′), GMR-Gal4 UAS-Yki; UAS-14-3-3ζ RNAi/UAS-HA-14-3-3ζ(H and H′), GMR-Gal4 UAS-Yki (I and I′), GMR-Gal4 UAS-Yki; 14-3-3εj2B10/+ (J and J′), GMR-Gal4 UAS-Yki; 14-3-3ζ12BL/+ (K and K′) and GMR-Gal4 UAS-Yki; 14-3-3εj2B10/+ 14-3-3ζ12BL/+ (L and L′). Reduction of 14-3-3 either by RNAi knockdown or loss of function mutations enhanced the overgrown phenotype caused by Yki overexpression.
Figure 2
Figure 2. Overexpression of either 14-3-3 isoform can suppress Yki-induced eye overgrowth
(A-E) Side views (A-E) or dorsal views (A′-E′) of adult eyes of GMR-Gal4 (A and A′), GMR-Gal4 UAS-Yki (B and B′), GMR-Gal4 UAS-Yki; UAS-HA-14-3-3ε (C and C′), GMR-Gal4 UAS-Yki; UAS-HA-14-3-3ζ(D and D′), GMR-Gal4 UAS-Yki; UAS-HA-14-3-3ε+ UAS-HA-14-3-3ζ(E and E′) and GMR-Gal4; UAS-HA-14-3-3ε+ UAS-HA-14-3-3ζ(F and F′).
Figure 3
Figure 3. Loss of 14-3-3 results in nuclear accumulation of Yki
(A and B) Low (A) and high (B) magnification view of a wild type wing disc immunostained with an anti-Yki (green) antibody. Yki is primarily localized in the cytoplasm. (C-D″) Low (C) and high (D-D″) magnification view of a wing disc expressing both UAS-14-3-3ε RNAi and UAS-14-3-3ζ RNAi together with UAS-P35 using MS1096 and immunostained with anti-Yki antibody (green in C, D and D″) and 7-AAD (red in D′ and D″) to label the nuclei. The wing disc is oriented with anterior to the left and ventral up. Severe knockdown of both 14-3-3 isoforms in the dorsal region of the wing pouch resulted in nuclear accumulation of endogenous Yki.
Figure 4
Figure 4. Multiple phosphorylation events contribute to Yki regulation
(A-D′) Side views (A-D) or dorsal views (A′-D′) of adult eyes expressing UAS-Myc-Yki (A and A′), UAS-Myc-YkiS168A (B and B′), UAS-Myc-YkiS111,250A (C and C′) or UAS-Myc-Yki3SA (D and D′) with GMR-Gal4. (E) S2 cells were transfected with the indicated Yki constructs together with Sd and an Sd-luciferase reporter gene, with or without Hpo and Wts expressing constructs. Cell lysates were subjected to dual luciferase assay. Error bars indicate standard deviation (triplicate wells). Numbers indicate degrees of suppression of Yki/Sd activity by Hpo/Wts.
Figure 5
Figure 5. Phosphorylation at S111 and S250 restricts Yki nuclear localization
(A-D′) S2 cells expressing HA-Sd together with Myc-YkiWT (A and A′), Myc-YkiS168A (B and B′), Myc-YkiS111,250A (C and C′) or Myc-Yki3SA (D and D′) with (A′-D′) or without Fg-Hpo (A-D) were immunostained with anti-Myc (green) and anti-HA (red) antibodies. (E-H) High-magnification views of wing discs expressing HA-Sd and Myc- YkiWT (E), Myc-YkiS168A(F), Myc-YkiS111,250A(G) or Myc-Yki3SA (H) using MS1096 and immunostained with anti-Myc (green) and anti-HA (red) antibodies.
Figure 6
Figure 6. Phosphorylation at S111 and S250 regulates Yki independent of 14-3-3
(A) The alignment of Yki sequences surrounding S111, S168 or S250 with the consensus sequence for Wts phosphorylation site indicated underneath. X: any amino acid. (B) Mutating S111 and S250 does not affect 14-3-3 binding but mutating S168 abolishes 14-3-3 binding. S2 cells were transfected with indicated Myc-tagged wild type and mutant Yki constructs and HA-14-3-3 constructs with or without Hpo/Wts/Mats coexpression, followed by immunoprecipitation and western blot analyses with indicated antibodies. (C) 14-3-3 regulates nuclear localization of YkiS111, 250A but not YkiS168A. S2 cells were transfected with HA-SD, Myc-YkiS111, 250A or Myc-YkiS168A, without or with coexpression of Hpo or Hpo plus 14-3-3. Cells were immunostained with Myc and HA antibodies, and the subcellular localization of Myc-YkiS111, 250A or Myc-YkiS168A was monitored using confocal microscopy. Cells with different nucleocytoplasmic distributions of Myc tagged Yki were counted. A total of 100 cells were counted for each Yki construct. C>N: cells contain higher levels of Yki in the cytoplasm than in the nucleus. C=N: cells contain Yki equally distributed in cytoplasm and nucleus. N>C: cells contain higher levels of Yki in the nucleus than in the cytoplasm. The y axis indicates the percentage of cells in each category. (D-G″) Side (top) or (bottom) dorsal views of adult eyes expressing GMR-Gal4 UAS-Yki (D), GMR-Gal4 UAS-YkiS111, 250A (E), GMR-Gal4 UAS-YkiS168A (F) or GMR-Gal4 UAS-Yki3SA (G). HpoN (D′-G′) or HpoN and 14-3-3 (D″-G″) were coexpressed with the wild type or mutant Yki as indicated.
Figure 7
Figure 7. Restriction of Yki nuclear localization by Hpo signaling depends on nuclear export
S2 cells were transfected with Myc-tagged wild type or mutant forms of Yki together with HA-Sd and with or without a Flag-Hpo expression construct. Cells were treated without or with LMB at a concentration of 20 nM for 4 hours, followed by immunostaining with Myc and HA antibodies. For each Yki construct, 100 cells were randomly selected and Yki subcellular localization was determined using confocal microscopy. C>N: cells contain higher levels of Yki in the cytoplasm than in the nucleus. C=N: cells contain Yki equally distributed in cytoplasm and nucleus. N>C: cells contain higher levels of Yki in the nucleus than in the cytoplasm.
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